Properties of Filament-bound Myosin Light Chain Kinase

Abstract

Myosin light chain kinase binds to actin-containing filaments from cells with a greater affinity than to Factin. However, it is not known if this binding in cells is regulated by Ca 2؉ /calmodulin as it is with F-actin. Therefore, the binding properties of the kinase to stress fibers were examined in smooth muscle-derived A7r5 cells. Full-length myosin light chain kinase or a truncation mutant lacking residues 2-142 was expressed as chimeras containing green fluorescent protein at the C terminus. In intact cells, the full-length kinase bound to stress fibers, whereas the truncated kinase showed diffuse fluorescence in the cytoplasm. After permeabilization with saponin, the fluorescence from the truncated kinase disappeared, whereas the fluorescence of the full-length kinase was retained on stress fibers. Measurements of fluorescence intensities and fluorescence recovery after photobleaching of the full-length myosin light chain kinase in saponin-permeable cells showed that Ca 2؉ /calmodulin did not dissociate the kinase from these filaments. However, the filament-bound kinase was sufficient for Ca 2؉ -dependent phosphorylation of myosin regulatory light chain and contraction of stress fibers. Thus, dissociation of myosin light chain kinase from actin-containing thin filaments is not necessary for phosphorylation of myosin light chain in thick filaments. We note that the distance between the N terminus and the catalytic core of the kinase is sufficient to span the distance between thin and thick filaments.

Deletion of MLCK210 induces subtle changes in vascular reactivity but does not affect cardiac function

“…Hence the effects related to the deletion of MLCK210 might be the consequence of either the absence of its kinase activity or of the loss of structural binding with protein partners. Because the pharmacological inhibition of the catalytic domain of MLCK in WT mice reproduced the same pattern of decreased shear stress as MLCK210 gene deletion, it suggests that the kinase activity of MLCK is the main contributor to this response rather than direct interactions between MLCK and other proteins (16,17,23). Moreover, the relative rapid time course of the "knockout-like" effect of the inhibitor (1 h) may point out that the alterations seen in the KO are not the result of chronic adaptation and are mimicked by the acute loss of enzymatic activity.…”

“…Of note, both isoforms are present in mature endothelial cells (4). These two MLCK isoforms differ in the length of their NH 2 -terminal tail domain, which contains amino acid sequence motifs associated with subcellular targeting or protein-protein interactions in the proteome (17,23). This noncatalytic domain of MLCK is distinct from the kinase domain.…”

KCNQ1 Channels Voltage Dependence through a Voltage-dependent Binding of the S4-S5 Linker to the Pore Domain

“…One possibility is that the cytosol provides an environment to solubilize the peptide in the millimolar range and that this range must be reached to uncover the peptide-peptide or peptide-channel interaction. Importantly, proteins expressed under the control of a CMV promoter can reach expression levels in the millimolar range (27,28 (Figs. 4 and 5), the S4S5 L /S6 T peptide interaction (Fig.…”

Microfilament-binding properties of N-terminal extension of the isoform of smooth muscle long myosin light chain kinase

“…In this extension region, there are six immunoglobin-like modules, a DVRXXL motif and a DFRXXL motif (usually called 2DFRXXL motif). There is evidence that demonstrated the importance of both 5 DFRXXL of long MLCK and 3 DFRXXL of short MLCK in F-actin binding [20,21]; each DFRXXL may bind to a single actin monomer in an actin filament [22]. Both 5 DFRXXL and 3 DFRXXL are sufficient for MLCK's binding to actin filament with maximal binding stoichiometries [23].…”

“…Fluorescence imaging of transfected NIH3T3 cells with different MLCK-GFP variant constructs was performed using a fluorescence microscope as described previously [21,23]. Twelve-bit images were obtained with a CCD camera (Quantix Photometrics; Tucson, AZ) and Oncorimage software (Oncor; Gaithersburg, MD).…”